Copper complexes of alpha-amino acids that contain terminal amino groups, and their use as nutritional supplements

ABSTRACT

In solid form copper complex salts of the formula: ##STR1## wherein &#34;n&#34; is from 1 to 5, and &#34;Z&#34; is an anion and &#34;y&#34; is the number required to electrostatically balance the set.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of earlier filed copendingapplication Ser. No. 285,593, filed Dec. 16, 1988.

BACKGROUND OF THE INVENTION

The important physiological role of copper in vertebrates was firstdemonstrated in 1928. Since that time copper was found to be a keycomponent of several enzymes. Most of these copper-containing enzymesare known to catalyze physiologically important reactions. Dietarydeficiency of copper in vertebrates results in several pathologicaldisorders.

One early manifestation of copper deficiency is spontaneous fractures ofbones in sheep and cattle that graze in copper-deficient pastures.Experimentally induced copper deficiency resulted in bone abnormalitiesin rabbits, pigs, chickens and dogs. The bone defect appears to residein the organic matrix and not in the mineral make-up of the bone.

Copper deficiency results in the failure of pigmentation of hair andwool in numerous species including black-wooled sheep in which bands ofpigmented and unpigmented wool can be correlated with coppersupplementation. Feathers of turkey poults fed copper-deficient dietsalso show lack of pigmentation.

Copper deficiency can cause cardiac lesion in cattle which may evenresult in sudden death. Further, chicks and pigs fed copper-deficientdiets may die suddenly from massive internal hemorrhage due tostructural defects in major arteries.

Subnormal levels of copper in the forage has resulted in neonatal ataxiaor swayback in lambs. Copper-deficient pigs do not absorb iron at anormal rate. It appears that copper is also essential for the release ofiron from the intestinal mucosa and iron storage tissues. In general,copper deficiencies cause anemia in all the species that have beenstudied so far.

Only a fraction of the copper ingested in diet is ultimately utilized inthe formation of copper-proteins. The form of the copper ingestedinfluences the degree of its utilization as measured by ceruloplasmin(copperprotein enzyme) activity. The presence of some amino acids havebeen known to cause an increase in the absorption and utilization ofcopper. (Kirchgessner and Girassmann, Z. Tierphysiol. TierenahrungFuttermittelk. 26:3 (1970); Girassmann et al., Z. Tierphysiol.Tierernahrung Futtermittelk. 28:28 (1971); Schwarz et al., ibid. 31:98(1973).)

It can therefore be seen that proper dietary balance of highlybioavailable copper is important for animals, including swine andpoultry. In recent times, it has been reported, particularly for swineand poultry, that they may have copper deficiencies because of thenature of their food rations. Put another way, it has been found thatfrom time to time mycotoxins which are present in food rations,particularly corn, have a tendency to tie up the available copper in aform which makes it nonbioavailable. Thus, the net result is that thefeed for the swine and the poultry, containing mycotoxins may in someway bind the available copper in the food to make it non-bioavailable.The net result is that even though one feeds to the animals much higherlevels of copper than the NRC (National Research Council) recommendeddaily allowances, the animal does not get anywhere near its adequate NRCrequirement.

An additional problem is caused by feeding high levels of copper whichare simply thereafter excreted without uptake. The copper in theexcretement is returned to the soil, significantly raising the copperlevel. This may cause undesired environmental polluting problems.

It therefore can be seen that adequate dietary levels of highlybioavailable copper are necessary for development of livestock,including cattle, swine and poultry. Moreover, there is a need topresent copper in a highly bioavailable form which assures that theanimal will have a high level of uptake of the copper without excretingit to cause potential environmental pollution.

It is an especially preferred advantage to make the complexes of thepresent invention from essential amino acids. This allows not only forincreased bioavailability of the copper, but also it allows the animal aproper nutritional amount of an essential amino acid necessary forproper growth. For copper, it is especially difficult to form solid formcomplexes of copper and alpha-amino acids. For example, if one attemptsto form such a complex with methionine in accord with the generalprocedures outlined in U.S. Pat. No. 3,941,818 issued Mar. 2, 1976 andU.S. Pat. No. 4,021,569 issued May 3, 1977, the result is anoxidation-reduction reaction between the methionine and the copper,resulting in an unuseable reaction product. It has now been discoveredthat when copper complexes of alpha-amino acids having a terminal aminegroup are employed, the internal redox reaction does not occur.

It is therefore a primary objective of the present invention to providea highly effective bioavailable form of copper in convenient watersoluble salt form which is available for use as a feed additive inanimal nutrition.

Another important objective of the present invention is the preparationof new, complex salts of copper in which the copper is in a form thatcan be readily absorbed after ingestion by livestock, particularlyswine, poultry and cattle.

Yet another objective of the present invention is to provide complexedsalts of copper which, because of the complexing of copper with certainalpha-amino acids containing a terminal amine moiety are shelf stable.

An even further objective of the present invention is to provide amethod of nutritional supplementation for animals to assure adequatedietary requirements of copper for growth and health.

An even further objective of the present invention is to provide certaincomplex salts of copper and certain alpha-amino acids containing aterminal amine moiety, such as lysine, which have coordination bondsformed between the copper ion and the alpha-amino group of the acid, inaddition to an electrostatic attraction bond between the cation and thecarboxyl ions.

The method of accomplishing these as well as other objectives of theinvention will become apparent from the detailed description of theinvention which follows hereinafter.

SUMMARY OF THE INVENTION

This invention relates to the preparation of copper complexes having thefollowing general formula: ##STR2## wherein "n" is from 1 to 5, and "Z"is an anion and "y" is the number required to electrostatically balancethe set. Because of a complex formed between the copper and thealpha-amino acid, it is believed these compounds are in a form that isreadily absorbed by domestic livestock, including cattle, swine, andpoultry. These complexes thus function as a readily available source ofcopper for dietary supplementary while simultaneously providingessential alpha-amino acids for normal growth and health.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention can be described as coppercomplex salts between the copper cation and certain alpha-amino acidswith a terminal amine moiety of the general formula: ##STR3## wherein"n" equals 1-5. The preferred acid is when "n" equals 4, lysine.However, "n" should not increase substantially beyond 5 becausecompounds for "n" is greater than 5 are not readily available and notcommercially feasible.

It is also important to note that the compounds of this invention arecopper complexes in which coordination bonds are formed between thecopper cation and the alphaamino group of the acid, in addition to theelectrostatic attraction between the cation and the carboxyl ions. Thiscomplex salt involving both coordination bonds and electrostaticattraction seems to enhance the bioavailability of the copper. Thegeneral formula of these complexes, illustrating both the coordinationbond and the electrostatic attraction, can be represented as follows:##STR4##

While the above formula represents the compounds sterically, thecompounds can be represented as well by the following formula: ##STR5##"n" is 1 to 5, and "Z" is an anion, and "y" is the number required toelectrostatically balance the salt. It is important to remember thatthese salts are capable of existing in stable solid state form. They arethus processible and useable in animal feeds. This is to bedistinguished from mere existence in non-isolatable transient states inan equilibrium of a solution. Preferred salts are those of thealpha-amino acid lysine wherein "n" equals 4. The most preferred salt iscopper lysine sulfate. In the formula presented, "Z" equals the anion,and may be an inorganic anion or an organic anion. Preferably, "Z" isselected from the group consisting of chloride, bromide, iodide,sulfates and phosphates. By the term "sulfates" and "phosphates" it isintended to include acid sulfates and acid phosphates, as well as thosehaving no hydrogen moieties associated with either the sulfate or thephosphate, i.e. simply the sulfate SO₄ ⁻⁻ and the phosphate PO₄ ⁻³.

For the preferred solid form copper complexes of lysine and copperchloride, or copper sulfate, the complexes are stable, very watersoluble, blue crystalline materials.

For details of other copper complexes of the hydroxy acids for similaruses, see co-pending and commonly assigned application for COPPERCOMPLEXES OF ALPHA HYDROXY ORGANIC ACIDS AND THEIR USE AS NUTRITIONALSUPPLEMENTS, Ser. No. 260,023, filed Oct. 20, 1988.

Simple and economically feasible processes of preparing these coppercomplexes have been developed. In accord with the process of thisinvention, a solution of the lysine monohydrate is heated to 60°-80° C.Hydrochloric acid is carefully added followed by the careful addition ofcopper oxide. The mixture is stirred for approximately 30 minutes. Thesmall amount of insoluble copper oxide is removed by filtration. Thefiltrate is concentrated to dryness using conventional methods, such ashot air oven, spray drying, freeze drying or evaporation under reducedpressure, to give the desired product.

The same products may be obtained by carefully mixing a solution oflysine monochloride with basic cupric carbonate. The mixture is heatedunder reflux for two hours. The solution was filtered while hot andcooled to 20° C. Acetone is added step-wise to the cooled filtrate untilturbidity is observed. The mixture is stored overnight at 4° C. toprecipitate the desired product. The desired product may also beobtained from the filtrate by using a conventional drying method. Thedesired product could be further purified by recrystallization fromacetone-water.

Another convenient method for obtaining the copper complexes involvesthe careful addition of powdered copper sulfate to a hot solution oflysine monohydrate. The desired product is obtained from the mixture asdescribed in the previous examples.

The formation of copper complexes of basic alpha-amino acids such aslysine, Ornithine and gamma amino butyric acid are commonly used as ameans of protecting or blocking the alpha-amino group in syntheticreactions in which it is desired to derivatize the side chain aminogroup. (Ref. A. C. Kurtz, J. Biol. Chem., 180, 1253 (1949); R. Roeskeand F. H. C. Stewart, R. J. Stedman and Vincent Du Vigneaud, J. AmericanChemical Society, 78, 5883 (1956). However, the procedures used in thereactions involve small amounts of reactants and complex methods whichare not applicable for the preparation of large amounts of product.Additionally, the copper complexes are not usually isolated incrystalline form and are not characterized.

A distinct advantage of the compounds of the present invention is thatthey are crystalline, stable and water soluble compounds which can beeasily obtained in large quantities for use as feed additives at arelatively low cost. The compounds of this invention provide a readilyavailable source of copper and the essential alpha-amino acid lysine.

The level of addition of the copper alpha-amino acids containing aterminal amino moiety of this invention for use as a feed supplement canvary over a wide range. Preferably the level of addition is such toprovide dietary intake of copper from about 0.2 parts per million toabout 2.0 parts per million, and most preferably about 1.0 parts permillion. These levels are satisfactorily achieved when the amount of thepreferred complex salt of copper lysine as well as others, added to theanimal feed, is from about 0.1 g per head of cattle per day to about 1.0g per head of cattle per day, preferably 0.6 g per head of cattle perday. It should, however, be understood that other levels of addition canbe utilized and that the precise level of addition is not in factcritical, it being adjusted for the conditions of the animals beingtreated with the nutritional supplement.

Importantly, one of the distinct advantages of the compounds of thepresent invention is that they are crystalline water soluble compoundseasily processible and size reducible to a powder for use in aconvenient feed supplement form. In other words, their physical form isone which is easily mixable with typical ruminant animal feeds.

In making nutritional supplements for addition to the diets of animals,it is preferred that the complexes of the present invention be added tocarrier or filler materials for processibility, ease of handling andsale. Examples of suitable carriers include distillers fermentationsolubles, feed grains, animal, poultry and fish bi-products and meal,whey and other cellulosic carrier materials well-known in the tracemineral product preparation techniques of the art.

The folowing example is offered to illustrate, but not limit, thepreparation of the compounds of the present invention.

EXAMPLE 1

Basic cupric carbonate (12 gm.) was added portionwise very carefully toa solution of lysine monochloride (10 gm.) in water (100 ml.). Themixture was heated under reflux for 2 hours. The solution was filteredwhile hot. The filtrate was cooled to 20° C. and acetone was addedstepwise until turbidity was observed. The mixture was stored at 4° C.overnight. The precipitate was collected and dried. An analytical samplewas prepared by recrystallization from water-acetone. The sample wasdried over phosphorus pentoxide. The infrared spectrum of the productwas recorded in KBr pellet and found to be compatible with the structureherein given. Elemental analysis for C₁₂ H₂₈ O₄ N₄ C1₂ Cu, 2 H₂ Ocalculated found: C, 31.14 (31.51); H, 6.96 (6.93); N, 12.10 (11.86);C1, 15.31 (15.15). Copper analysis using ashing procedure. Ash 14.33percent; copper calculated at 11.44 percent from ash. Theory was 13.73percent. This elemental analysis confirms the structure.

EXAMPLE 2

Lysine monohydrate (16.7 gm.) was mixed with water (25 ml.) and themixture was heated with stirring at 60°-80° C. until all the solid wasdissolved. Concentrated hydrochloric acid (9.8 ml. of 37.3 percent) wasadded very carefully. The stirring of the solution was continued duringthe addition of acid. Copper oxide (4.0 gm.) was added to the stirredsolution and stirring was continued at 70° C. for 30 minutes. The hotmixture was filtered to remove unreacted copper oxide. The filtrate wasconcentrated to dryness to give the desired product (21 gm.). Theproduct is very soluble in water and gives a clear deep blue solution.The color of the solid product depends on the degree of hydration. Ananhydrous product has a yellow-green color, as the solid becomeshydrated it changes to deep blue. The solid has the same chemical andphysical properties as that of the product obtained in Example 1.

EXAMPLE 3

Lysine monohydrate (800 pounds) was mixed with 1,400 pounds of hotwater. The solution was mixed until all the lysine had dissolved(approximately 10 minutes). Next, stirring was continued while coppersulfate pentahydrate (600 pounds) was slowly added.

After all the copper sulfate pentahydrate had been added to the lysinesolution, stirring was continued and the solution was maintained at160°-180° F. for 10-15 minutes to allow the complexing reaction tooccur.

The copper lysine complex was dried using a conventional spray drytechnique. The resulting desired product was a green fine powder.

This compound, if fed to livestock, particularly swine, cattle andpoultry and also for example sheep at the levels specified herein, willshow that the copper is in a highly bioavailable form and will provideit at levels sufficient for adequate dietary intake of copper to providenormal healthy growth, good red blood cell production, properpigmentation for hair and wool, and at sufficient levels for normalreproductive patterns.

It therefore can be seen that the invention does and will accomplish allof its stated objectives.

What is claimed is: 1.In solid form, copper complex salts of theformula: ##STR6## wherein "n" is from 1 to 5, and "Z" is an inorganicanion and "Y" is the number required to electrostatically balance thesalt.
 2. The salts of claim 1 wherein said salt is in a finely dividedstate, suitable for use as a feed supplement.
 3. The copper complexes ofclaim 1 wherein "n" is
 1. 4. The copper complexes of claim 1 wherein "Z"is selected from the group consisting of chloride, bromide, iodide,sulfates and phosphates.
 5. A method of assuring adequate dietaryrequirements of bioavailable copper for good growth and yield productionof livestock, said method comprising:adding as a feed ration supplementto the animal's feed,a small but effective amount of a copper complexsalt of the formula: ##STR7## wherein "n" is from 1 to 5, and "Z" is ananion and "y" is the number required to electrostatically balance theset.
 6. The method of claim 5 wherein the amount of said salt added tothe animal's diet is from 0.1 to 1.0 g per head per day.
 7. Copperlysine sulfate.